title: CLIMP-63 is a gentamicin-binding protein that is involved in drug-induced cytotoxicity.
abstract: Aminoglycoside-induced nephrotoxicity and ototoxicity is a major clinical problem. To understand how aminoglycosides, including gentamicin, induce cytotoxicity in the kidney proximal tubule and the inner ear, we identified gentamicin-binding proteins (GBPs) from mouse kidney cells by pulling down GBPs with gentamicin-agarose conjugates and mass spectrometric analysis. Among several GBPs specific to kidney proximal tubule cells, cytoskeleton-linking membrane protein of 63 kDa (CLIMP-63) was the only protein localized in the endoplasmic reticulum, and was co-localized with gentamicin-Texas Red (GTTR) conjugate after cells were treated with GTTR for 1 h. In western blots, kidney proximal tubule cells and cochlear cells, but not kidney distal tubule cells, exhibited a dithiothreitol (DTT)-resistant dimer band of CLIMP-63. Gentamicin treatment increased the presence of DTT-resistant CLIMP-63 dimers in both kidney proximal (KPT11) and distal (KDT3) tubule cells. Transfection of wild-type and mutant CLIMP-63 into 293T cells showed that the gentamicin-dependent dimerization requires CLIMP-63 palmitoylation. CLIMP-63 siRNA transfection enhanced cellular resistance to gentamicin-induced toxicity, which involves apoptosis, in KPT11 cells. Thus, the dimerization of CLIMP-63 is likely an early step in aminoglycoside-induced cytotoxicity in the kidney and cochlea. Gentamicin also enhanced the binding between CLIMP-63 and 14-3-3 proteins, and we also identified that 14-3-3 proteins are involved in gentamicin-induced cytotoxicity, likely by binding to CLIMP-63.
authors.affiliation: Oregon Hearing Research Center, Oregon Health & Science University, Portland, 97239, USA. karasawa@ohsu.edu
authors.forename: T
authors.initials: T
authors.lastname: Karasawa
authors.forename: Q
authors.initials: Q
authors.lastname: Wang
authors.forename: L L
authors.initials: LL
authors.lastname: David
authors.forename: P S
authors.initials: PS
authors.lastname: Steyger
mesh_terms: D048948:14-3-3 Proteins / Q000378:metabolism; D000818:Animals; D000900:Anti-Bacterial Agents / Q000633:toxicity*; D002478:Cells, Cultured; D003051:Cochlea / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D019281:Dimerization; D004229:Dithiothreitol / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005839:Gentamicins / Q000633:toxicity*; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D054878:Lipoylation; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D011485:Protein Binding; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D014966:Xanthenes / Q000737:chemistry
chemical_list: D048948:14-3-3 Proteins; D000900:Anti-Bacterial Agents; C558686:CLIMP-63 protein, mouse; D005839:Gentamicins; D008565:Membrane Proteins; D034741:RNA, Small Interfering; D014966:Xanthenes; C034657:Texas red; D004229:Dithiothreitol